Part:BBa_K2889003:Experience

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Applications of BBa_K2889003

We constructed two plasmids pEGFP-miR-21-sponge-4s and PSB1C3-miR-21-sponge-4s. The plasmids of pEGFP-miR-21-sponge-4s and PSB1C3-miR-21-sponge-4s were used to monitor the expression of miR-21 in cells. And the plasmid of pSB1C3-miR-21-sponge-4s will be submitted to 2018IGEM.

In our previous project, we constructed two miR-21 sponge plasmid, pEGFP-miR-21-sponge-2s and pEGFP-miR-21-sponge-6s.Subsequently, we got two standard curves by transfected pEGFP-miR-21-sponge-2s and pEGFP-miR-21-sponge-6s with different concentration of pHAGE-pre-miR-21 to 293T cells and calculated the copy numbers of miR-21.However, we’d like to ascertain a preferable binding sites number contained in miRNA sponge as a sensitivity and specificity monitor this year.Hence, we constructed pEGFP-miR-21-sponge-4s.Firstly, we designed miR-21 sponges contains four miR-21 binding sites with 3-nt spacers for bulged sites based on the sequence of hsa-miR-21 according to the previ-ous study.Then we amplified miR-21-sponge-4s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes Xho I and Hind III. Finally, miR-21-sponge-4s fragments was ligated to pEGFP-c1 vector.To test the effect of miR-21 sponges as a monitor to detect the expression of miR-21, 293T cells were transfected with pEGFP-miR-21-sponge-4s with different concentra-tion of pHAGE-pre-miR-21. We used the plasmid of pHAGE-pre-miR-21 to quantify miR-21 expression and calculate the copy numbers of miR-21 by using the formula.Finally,We find that miR-21-sponge-4s is more sensitive than miR-21-sponge-2s and miR-21-sponge6s.

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